Pheromones_Male_Lures_and_Trapping_of_Te.pdf

Chapter 2
Pheromones, Male Lures, and Trapping
of Tephritid Fruit Flies
Keng Hong Tan, Ritsuo Nishida, Eric B. Jang, and Todd E. Shelly
Abstract Both sex pheromones and male lures appear to play an important role in
the mating systems of many species of economically important tephritid species.
Typically, stationary males emit pheromone attractive to searching females, and
recent evidence indicates that naturally occurring male lures may function as pre-
cursors in pheromone synthesis. Here, we review (i) the basic biology of sex
pheromones and the importance of naturally occurring male lures as pheromone
components or precursors and (ii) the use of sex pheromones and male lures as trap
baits, primarily in fruit fly detection programs, for the major genera of Anastrepha,
Bactrocera, Ceratitis, Dacus, Rhagoletis, and Toxotrypana. Relatively few studies
have examined the effectiveness of pheromone-based trapping, and most of these
have involved only three species, the Mediterranean fruit fly, Ceratitis capitata
(Wiedemann), the Mexican fruit fly, Anastrepha ludens (Loew), and the Caribbean
fruit fly, A. suspensa (Loew). In general, the results have been inconsistent, with
traps baited with live males or male pheromone extracts or components attracting
more females than blanks or food-baited traps in some studies but not in others.
This inconsistency, along with the chemical complexity of pheromones and the
K.H. Tan (*)
Tan Hak Heng, 20, Jalan Tan Jit Seng, 11200 Tanjong Bungah, Penang, Malaysia
e-mail: tan.kenghong@yahoo.com
R. Nishida
Graduate School of Agriculture, Laboratory of Chemical Ecology, Pesticide Research
Institute, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
e-mail: tan.kenghong@yahoo.com
E.B. Jang
USDA-ARS, U.S. Pacific Basin Agricultural Research Center, 64 Nowelo Street, Hilo,
HI 96720, USA
e-mail: eric.jang@ars.usda.gov
T.E. Shelly
USDA-APHIS, 41-650 Ahiki Street, Waimanalo, HI 96795, USA
e-mail: todd.e.shelly@aphis.usda.gov
T. Shelly et al. (eds.), Trapping and the Detection, Control, and Regulation of Tephritid
Fruit Flies, DOI 10.1007/978-94-017-9193-9_2,
© Springer Science+Business Media Dordrecht 2014
15

multimodal nature of sexual communication (where olfaction is but one of several
sensory channels used in male signaling and courtship), has limited research on the
development of pheromone baits. Male lures, on the other hand, have proven
incredibly useful and consistently effective trap baits. The major male lures –
methyl eugenol, cue-lure/raspberry ketone, and trimedlure – are discussed as are
possible replacements/modifications, such as fluorinated analogues of methyl euge-
nol, raspberry ketone formate, zingerone, ceralure, and enriched ginger root oil. In
addition, we discuss various factors influencing the efficacy of male lures, including
fly age, prior lure ingestion, selection for non-responsiveness, interspecific differ-
ences in responsiveness, and the use of liquid versus solid dispensers.
Keywords Aggregation • Anisylacetone • Attractant • α-copaene • Cuelure •
Electroanntenogram • Floral volatile • Kairomone • Male lure • Methyl eugenol •
Orchid • Phenylbutanoid • Phenylpropanoid • Pheromone • Raspberry ketone •
Rectal gland • Sesquiterpene • Synomone • Trimedlure
1 Introduction
Chemical cues and signals influence the behavior, physiology, and ecology of
insects in a remarkably large number of ways. It is hardly surprising, then, that
strategies designed to protect agricultural systems are often based on chemical
stimuli and cues important to pestiferous insects. These strategies are themselves
diverse and may involve the elimination, modification, disruption, imitation, or
circumvention of chemical information important to the target insect. Tephritid
fruit flies are trapped for a variety of reasons – surveillance, suppression, and
ecological study among others – and chemical baits have played a central role in
these efforts. The existence of male lures was reported approximately 100 years ago
(Howlett 1912, 1915), and such lures have been among the most widely used in
programs to detect and manage tephritid fruit fly pests. Likewise, the presence of
sex pheromones in economically important Tephritidae has been recognized for
over 50 years (Féron 1959), and though not yet as effective as male lures, they have
received considerable attention as possible tools in fruit fly surveillance and control.
This chapter provides an overview of the use of pheromones and male lures in
trapping economically important fruit flies of the genera Anastrepha, Bactrocera,
Ceratitis, Dacus, Rhagoletis, and Toxotrypana. Given the broad scope of this topic
and the accompanying rich body of literature, our review is not exhaustive. Though
somewhat idiosyncratic, reflecting invariably our own research experiences, we
nonetheless believe we have highlighted main themes and introduced some new
ideas or perspectives as well.
As evidenced by the chapter title, we have decided to describe compounds, such
as methyl eugenol, cue-lure, raspberry ketone, trimedlure, and others, as male lures
or male attractants and to avoid the oft-used term ‘parapheromone’. We do so for
three main reasons: (i) Payne et al. (1973) originally defined parapheromones as
16 K.H. Tan et al.

“compounds which are not naturally used in intraspecific insect communication”.
However, several studies (Nishida et al. 1988a, b, 1993; Tan and Nishida 1995,
2007; Tan et al. 2011) have demonstrated that certain male lures (e.g., methyl
eugenol, raspberry ketone, and zingerone) are used in synthesizing male sex
pheromones, and so the original definition of parapheromone does not apply to
tephritids; (ii) in a recent review of insect parapheromones, Renou and Guerrero
(2000) restrict parapaheromones to “chemical compounds of anthropogenic origin
not known to exist in nature”. Once again, this criterion does not apply to methyl
eugenol and raspberry ketone, which occur in many different plant species (Tan and
Nishida 1995, 2012), and so excludes these two important tephritid male attractants
(indeed, Renou and Guerrero’s review does not even include discussion of the
Tephritidae), and (iii) the very use of the root ‘pheromone’ implies that male lures
produce behavioral and/or physiological effects that resemble those of natural
pheromones. There is evidence that male-produced sex pheromones may attract
conspecific males and so act as aggregation pheromones (Nishida et al. 1988b; Tan
and Nishida 1996; Hee and Tan 1998; Khoo and Tan 2000; Wee and Tan 2005a;
Wee et al. 2007). Because male lures may (upon ingestion) be used in pheromone
synthesis (references above), the idea that the male lures mimic the male sex
pheromone appears reasonable and may eventually be shown to be valid. However,
the available data regarding male-male olfactory attraction derive exclusively from
laboratory studies (with a single exception, Nishida et al. 1988b). With few field
data available, we consider it premature to conclude that male lures resemble
pheromones in function. That said, we also recognize that the term male lure is
not completely accurate, since the lures are known to occasionally attract females
(Steiner et al. 1965; Nakagawa et al. 1970; Fitt 1981a; Verghese 1998). While not
dismissing the importance of these observations, our collective field experience
(except for a female Bactrocera umbrosa (Fabricius) captured by Tan in 2014) is
that males comprise the vast majority of all individuals observed at point sources
(traps, flowers, etc.) of known male lures, and hence the terms male lure or male
attractant are generally, if not always, appropriate.
2 Tephritid Pheromones and Trapping
The family Tephritidae contains several genera, namely Anastrepha, Bactrocera,
Ceratitis, Dacus, Rhagoletis and Toxotrypana, with species that are major agricul-
tural pests of fruits and vegetables. Information on the identification of pheromones
and their possible use in trapping programs is summarized below for each of these
genera.
2 Pheromones, Male Lures, and Trapping of Tephritid Fruit Flies 17

2.1 Anastrepha Pheromones
The genus Anastrepha contains approximately 200 species distributed in tropical
and subtropical areas of the New World (Norrbom et al. 2000) of which eight
(A. distincta Greene – Inga fruit fly, A. fraterculus (Wiedemann) – South American
fruit fly, A. grandis (Macquart) – South American cucurbit fruit fly, A. ludens
(Loew) – Mexican fruit fly, A. obliqua (Macquart) – West Indian fruit fly,
A. serpentina (Macquart) – sapote fruit fly, A. striata Schiner – guava fruit fly,
and A. suspensa (Loew) – Carribean fruit fly) are major agricultural pests, attacking
a wide variety of fruits and vegetables (Aluja 1994; Norrbom et al. 2012). Although
field observations are incomplete, many of the polyphagous and economically
important species appear to display a lek mating system in which males occupy
mating territories on leaves and attract females to the territory via a complex suite
of visual, acoustic, and olfactory signals (Aluja et al. 2000). Regarding the latter,
pheromone-calling males emit volatiles from everted pleural pouches and anal
membranes, with aerial dispersion aided by intense bouts of rapid wing vibrations
(Nation 1972). Volatile components are also released via the mouth (Nation 1990),
and abdominal dipping of the evaginated anal membranes to the leaf surface may
amplify pheromone attractiveness by increasing the evaporative surface area of the
volatile components (Sivinski et al. 1994). Pheromone calling has been observed
for a small number of Anastrepha species, and chemical analysis and identification
of pheromonal components has been undertaken for only a subset of these species
(Fig. 2.1 and Table 2.1).
Measurements of female attraction to male sex pheromone, or components
thereof, have been made for an even smaller subset of all species, with nearly all
of the research conducted on A. suspensa (Table 2.2) or A. ludens (Table 2.3). The
biological activity of male pheromones has been studied in only four other species,
with only one study undertaken for each. In both A. fraterculus and A. obliqua,
freshly dissected salivary glands of males were found to attract mature and virgin
females in laboratory cage tests (Lima et al. 2001; Ibañez-López and Cruz-López
2001). However, in A. serpentina, three putative pheromonal components were
examined, with no strong female response observed for any of them (Robacker
et al. 2009a, b). In Anastrepha sororcula Zucchi, field tests found no difference in
female captures in traps baited with live calling males versus blank control traps
(Santos Felix et al. 2009).
Despite the large amount of research conducted on A. suspensa and A. ludens,
trapping and detection efforts for these two species still rely primarily on food-
based lures (e.g., Robacker and Thomas 2007; Epsky et al. 2011), and an effective
pheromone-based trap has not been developed. Several authors (Landolt and Heath
1996; Landolt and Averill 1999) have enumerated the reasons for this, and these
generally include:
First, the long-range attractiveness of the male sex pheromone has weak empir-
ical support, since the majority of research has been conducted in the small,
laboratory cages and thus measures only short-range attractiveness or arrestant
18 K.H. Tan et al.

properties of the test chemicals. The widely used cage-top bioassay, for example,
has generally been used in cubical cages only (0.2–0.3 m per side). Moreover,
although the cage-top assay has generally indicated female response to male-
derived chemicals, other laboratory tests involving slightly larger cages have failed
to demonstrate long-range attraction of females to male pheromone. For example,
arrivals of A. ludens females did not differ significantly between citrus trees having
chemically-treated (with male pheromone extract or pheromonal components) or
control (blank) leaves (Robacker and Hart 1986; Robacker 1988). However, using a
1.2 m long wind tunnel and a videotape system, Heath et al. (1993) found that
A. suspensa females land more frequently on male-baited traps than control traps
but spent equal amounts of time on the two trap types after landing. These data
clearly indicate that the male volatiles are an attractant and not a simple arrestant. In
sum, then, use of small cages does not allow rigorous identification of long-range
attraction of Anastrepha females, and other laboratory results from slightly larger
cages are inconsistent in this regard.
Second, field tests have yielded inconsistent results regarding female attraction
to male-produced odors. In an early study, sticky traps baited with aggregations of
20 or 40 males captured significantly more released virgin females of A. suspensa
than McPhail traps baited with an aqueous solution of yeast hydrolysate (Perdomo
et al. 1975). A follow-up study on the same species (Perdomo et al. 1976) generated
the same result and also documented attraction of released males to the male-baited
traps. In contrast, although the difference was not statistically significant, Robacker
and Wolfenbarger (1988) found that food-baited McPhail traps captured three times
as many A. ludens females as pheromonal traps (baited with extracts of male
abdomens). Similarly, and as noted previously, field tests involving A. sororcula
found no difference in female captures in traps baited with live calling males versus
O
O
H
O
O
H
O
O
Anastrephin (E,E)-Suspensolide
(E,E)-a-Farnesene
Epianastrephin
Limonene
(Z,E)-a-Farnesene
(Z)-b-Ocimene
Fig. 2.1 Some sex pheromonal components of Anastrepha (Adapted from Rocca et al. 1992)

Table 2.1 Anastrepha species for which male pheromone calling has been observed and the
incidence of chemical analyses of pheromonal components in these species
Species
Pheromone
calling References
Pheromone
Chemistry References
A. bistrigata Bezzi + Da Silva et al. (1985) – –
A. fraterculus
(Wiedemann)
+ Malavasi et al. (1983),
Morgante
et al. (1983), Lima
et al. (1994), Segura
et al. (2007)
+ Cáceres et al. (2009),
Lima et al. (1996),
(2001)
A. ludens (Loew) + Aluja et al. (1983),
(1989), Moreno
et al. (1991),
Robacker
et al. (1991), (2003),
Robacker and Hart
(1985a), Aluja
et al. (2008)
+ Battiste et al. (1983),
Stokes et al. (1983),
Robacker and Hart
(1985b), Rocca
et al. (1992), Baker
and Heath (1993)
A. obliqua
(Macquart)
+ Aluja et al. (1983),
(1989) Da Silva
et al. (1985),
Meza-Hernández
et al. (2002), López
Guillén
et al. (2008),
Henning and
Matioli (2006)
+ Meza-Hernández
et al. (2002), Ibañez-
López and Cruz-
López (2001), López-
Guillén et al. (2008)
A. pseudoparallela
(Loew)
+ Da Silva et al. (1985),
Polloni and Da
Silva (1986)
– –
A. robusta Greene + Aluja (1993) – –
A. serpentina
(Macquart)
+ Aluja et al. (1989),
Castrejón -Gómez
et al. (2007),
Robacker
et al. (2009a)
+ Robacker et al. (2009a)
A. sororcula
Zucchi
+ Da Silva et al. (1985) – –
A. striata Schiner + Aluja et al. (1993),
(2008)
– –
A. suspensa (Loew) + Nation (1972), (1989),
(1990), Dodson
(1982), Burk
(1983), (1984),
Landolt and
Sivinski (1992),
+ Nation (1975), (1989),
(1990), (1991)),
Battiste et al. (1983),
Chuman et al. (1988),
Mori and Nakazono
(1988), Tumlinson
20 K.H. Tan et al.

Table 2.1 (continued)
Species
Pheromone
calling References
Pheromone
Chemistry References
Epsky and Heath
(1993a, b), Sivinski
et al. (1994)
(1988), Rocca
et al. (1992), Epsky
and Heath (1993a, b),
Baker and Heath
(1993), Heath
et al. (1993), Lu and
Teal (2001)
A. zenildae
(Zucchi)
+ De Almeida
et al. (2011)
– –
Table 2.2 Published accounts for Anastrepha suspensa regarding the behavioral response of
females to the sex pheromones of conspecific males
Odor source Bioassay arena
Positive response
Reference
Identified Observed
Live males Wind tunnel
(0.45 m long)
Move 25 cm
toward
source
Yes Nation (1972)
Pheromonal componentsa
Screen cage
(0.45 m per
side)
Enter trap Yes Nation (1975)
Live males Avocado grove Capture in male-
baited sticky
trapsb
Yes Webb
et al. (1983)
Pheromone extract Field cage Capture in sticky
traps
Yes Webb
et al. (1983)
Filter paper treated with
volatiles
Screen cage
(0.20 m per
side)
Aggregation near
treated paperc
Yes Sivinski and
Heath
(1988)
major pheromone
componentsd
Screen cage
(0.2 0.12
0.10 m)
Aggregation near
treated paperc
Yes Nation (1991)
minor pheromonal
componentse
Screen cage
(0.2 0.12
0.10 m)
Aggregation near
treated paperc
Nof
Nation (1991)
Live males Flight tunnel
(0.3 0.3
1.22 m)
Enter trap Yes Heath
et al. (1993)
a
Later identified (Nation 1983) as (Z)-3-nonenol, (Z,Z)-3; 6-nonadienol; anastrephin;
epianastrephin; attraction was observed for individual compounds as well as pairs and trios,
with greatest attraction observed for a combination of all four components
b
Released 13 m from any trap
c
A so-called cage-top test, where control and treated papers were placed, one per quadrant, placed
on top of cage, and distribution of females in four quadrants was measured
d
Same four as in footnote a
e
Bisabolene, ocimine, suspensolide
f
No response was observed when these compounds tested individually, but each increased female
response when added to blend containing the major components (listed in footnote a)

Table 2.3 Published accounts for Anastrepha ludens regarding the behavioral response of
females to the sex pheromones of conspecific males
Odor source Bioassay Arena
Positive response
Reference
Identified Observed?
Filter paper treated
with pheromone
extracta
Screen cage (0.3 m per
side)
Aggregation
near
treated
paperb
Yesc
Robacker and Hart
(1984), Robacker
et al. (1990),
Moreno
et al. (1991)
with pheromonal
componentsd
side)
Aggregation
near
treated
paperb
Ye
Robacker and Hart
(1985b)
Citrus leaf treated
with pheromonal
componentsf
Wind tunnel
(2 0.7 1.3 m)
Arrivals to
treated
leaves
Nog
Robacker (1988)
Citrus leaf treated
with combina-
tions of phero-
monal
componentsh
Wind tunnel
(2 0.7 1.3 m)
Arrivals to
treated
leaves
Yes Robacker (1988)
Citrus leaf treated
With pheromone
extracta
Wind tunnel
(2 0.7 1.3 m)
Arrivals to
treated
leaves
Yes Robacker (1988)
Citrus leaf treated
with pheromone
extracta
Screen cage
(0.7 1.6 1.0 m)
Arrivals to
treated
leaves
Yesi
Robacker and Hart
(1986)
Cigarette filter
treated with
pheromone
extracta
Citrus grove Capture in
treated
McPhail
traps
Yesj
Robacker and
Wolfenbarger
(1988)
with pheromone
extracta
Hallway
(30.0 2.5 2.0 m)
Upwind
move-
ment;
flightk
Yes Robacker and Moreno
(1988)
22 K.H. Tan et al.

blank control traps (Santos Felix et al. 2009). A field trial on the attractiveness of a
pheromonal blend likewise yielded negative results. In laboratory tests conducted
in small cages, A. suspensa females responded to four major components of the
male pheromone as well as various mixtures of these chemicals (Nation 1975; see
also Robacker and Hart 1985b for similar findings for A. ludens). However, a
synthetic blend of these same four components placed in the field failed to attract
flies of either sex over a 5-day period in Florida (Nation 1989). The uneven
performance of pheromone-baited traps in the field, coupled with data showing
that host fruit odors are equally or more attractive to females than male odors alone
(Robacker and Garcia 1990), has been an important constraint on further research
on the development of pheromone-based traps for Anastrepha.
Third, the pheromones of different Anastrepha males are complex and contain
multiple chemicals with different isomers (Heath et al. 2000). This complexity has
several important implications. It appears, for example, that the component ratios
Table 2.3 (continued)
Odor source Bioassay Arena
Positive response
Reference
Identified Observed?
Pheromone extractl
side)
Aggregation
near
treated
paperb
Yesm
Robacker and Garcia
(1990)
Live males Flight tunnel
(30 30 122 cm)
Enter trap Yes Heath et al. (1993)
a
Obtained from filtering and concentrating extract obtained from grinding abdomens of adult
males
b
A so-called cage-top test, where control and treated papers were placed, one per quadrant, placed
on top of cage, and distribution of females in four quadrants was measure
c
Attraction much stronger for mature virgin females than immature or recently mated females
d
Six components were tested individually and in various combinations: (Z)-3-nonenol, (Z,Z)-3,6-
nonadienol, (R,R)-(+)-anastrephin, (S,S)-( )-anastrephin, (R,R)-(+)-epianastrephin, (S,S)-( )-
epianastrephin
e
Only three components (the two alcohols plus epianastrephin) elicited female responses individ-
ually. Both synergistic and inhibitory effects were reported among the 15 combinations of paired
components
f
The six components listed in footnote d were tested individually
g
With a single exception: (Z,Z)-3,6-nonadienol attracted more females than control (untreated)
leaves
h
Three combinations involving pheromonal components listed in footnote d were tested: (Z)-3-
nonenol + (S,S)-( )-epianastrephin; (Z,Z)-3,6-nonadienol + (S,S)-( )-epianastrephin; all 6
components
i
Females did not distinguish between treated and control trees but within trees were more attracted
to treated than control leaves
j
Highest dose did not attract more females than control suggesting an overdose effect; male
attraction also observed
k
Females behavior monitored in screen cages placed at different distances from odor source, with
upwind movement scored as the number of females on upwind versus downwind sides of cages
l
Obtained from crushing whole males
m
Host fruit odor inhibited attraction of mature virgin females

affect the attractiveness of the blend. Differences in the attractiveness of two
dispensers to A. ludens females, for example, apparently reflected differential
release of pheromone components, which resulted in the emission of abnormal
component ratios for one of the dispensers (Robacker and Wolfenbarger 1988). In
general, data support the conclusion that individual pheromonal components stim-
ulate little behavioral response but instead function as an integrated unit to elicit
behavior (Robacker 1988), and identifying the specific nature of this complex
signal is seen as a daunting challenge. Moreover, the composition of male phero-
mone may vary with time of day (Tumlinson 1988; Nation 1990), social context
(calling singly or in a group, Nation 1990), and food availability (Epsky and Heath
1993a), making it even more difficult to identify the particular blend most attractive
to females. Analogously, variability in pheromone release rate (Epsky and Heath
1993b; Meza-Hernández et al. 2002) confounds identification of those rates that
may be maximally attractive to females. In addition, the different components have
different volatilities (Landolt and Averill 1999) and liabilities (Robacker
et al. 2009b), which render production of synthetic pheromones difficult from a
methodological perspective and imprecise from a biological one.
Finally, the importance of male pheromone to female mate searching remains
uncertain, and it appears likely that a combination of visual, auditory, and olfactory
cues may be involved. The pheromone appears to attract females to the vicinity of
calling males but not to point sources (Robacker 1988), and after approach, females
may rely on acoustic and/or visual signals to locate males (Webb et al. 1983;
Sivinski and Calkins 1986). As with the complex pheromonal blend, the multi-
faceted nature of mate location appears to have lessened the impetus to develop
pheromone-based traps for Anastrepha.
2.2 Bactrocera Pheromones
The genus Bactrocera consists of over 500 species distributed in the tropical and
subtropical regions of Asia (Smith et al. 2003) and includes many serious and/or
highly invasive polyphagus pest species, namely B. correcta (Bezzi) – guava fruit
fly, B. cucurbitae (Coquillett) – melon fly, B. carambolae Drew Hancock –
carambola fruit fly, B. dorsalis sensu stricto (Hendel) – oriental fruit fly, B.
invadens Drew, Tsuruta White, B. papayae Drew Hancock – Asian papaya
fruit fly, B. philippinensis Drew Hancock – Philippines fruit fly, B. latifrons
(Hendel) – solanaeous fruit fly, B. tryoni (Froggatt) – Queensland fruit fly,
B. umbrosa (Fabricius) – Artocarpus or jack-fruit fly, and B. zonata (Saunders) –
peach fruit fly. Males of these species, with the exception of B. cucurbitae and
B. tryoni (both attracted to cue-lure (CL)/raspberry ketone (RK)) and B. latifrons
(not attracted to either CL/RK or methyl eugenol (ME), are attracted to ME, a
compound found in a wide diversity of plant species (Tan and Nishida 2012) and
now known to be a pheromonal precursor. As discussed below, the strong attraction
of males to ME has, to some degree, limited impetus to explore sex pheromones as a
24 K.H. Tan et al.

trapping tool for Bactrocera species. Here, we summarize the chemistry of
Bactrocera pheromones and note studies that have monitored male or female
attraction to pheromonal emissions.
As true for most of the economically important tephritid species examined thus
far, sexual signaling in Bactrocera typically involves the production and broadcast
of sex pheromone by males (a behavior termed “calling”) while resting on vegeta-
tion and detection and subsequent mate searching by receptive females. Most
accounts of male calling and mating derive from laboratory or field cage observa-
tions (e.g., Tychsen 1977; Ohinata et al. 1982; Arakaki et al. 1984; Kuba and
Koyama 1985), and the few field studies conducted – all on B. dorsalis– indicate
plasticity in that species’ mating system. Working in Hawaii, Shelly and Kaneshiro
(1991) observed calling males and matings in the canopy of a single fruiting tree
within a citrus orchard, suggestive of a lek mating system. In contrast, Stark (1995),
also working in Hawaii, observed B. dorsalis females moving from papaya trees to
non-host (Panax) trees in the late afternoon followed by males 30–60 min later.
Although their incidence was not quantified, Stark (1995) observed matings on this
nonhost plant. Finally, working in Thailand, Prokopy et al. (1996) released
B. dorsalis within a non-fruiting orchard and experimentally added food, water,
and host fruits to the trees. In this case, and in contrast to the aforementioned
studies, all sexual behavior and all matings were recorded on trees with fruits and
on the fruit itself, leading the authors to suggest that the importance of host fruits as
foci for sexual activity may vary with microclimatic conditions. The behavioral
variability described for B. dorsalis, along with the lack of field studies on
Bactrocera species in general, serves as a cautionary prelude to the following
discussion: little is known about the importance of male pheromones in sexual
selection in the genus, and consequently evaluation of male pheromones as poten-
tial trap attractants is necessarily preliminary and inconclusive.
2.2.1 Sex Pheromone of B. dorsalis Complex Species
The B. dorsalis species complex comprises over 70 recognized species (White and
Elson-Harris 1992), several of which, namely B. dorsalis, B. invadens, B. papayae,
B. philippinensis, and B. carambolae, are serious agricultural pests. Recent molecular
(Tan et al. 2011, 2013; Schutze et al. 2012; Krosch et al. 2013), morphological
(Mahmood 1999, 2004; Schutze et al. 2012; Krosch et al. 2013), behavioral (i.e.,
mating compatibility; McInnis et al. 1999; Tan 2000, 2003; Wee and Tan 2005b;
Schutze et al. 2013), and pheromone chemistry (Tan and Nishida 1996, 1998; Tan
et al. 2011, 2013) data have raised doubts regarding the validity of species status for
these sibling taxa (except B. carambolae – see below). Below, we retain the names as
originally used but recognize that results obtained for one species may, if taxonomic
synonymies are eventually recognized (Schutze et al. 2014), apply to other currently
recognized species in the complex.
In the first published description on the pheromone chemistry of male Bactrocera,
Ohinata et al. (1982) analyzed “smoke” produced by male B. dorsalis and found that

trisodium phosphate was the major component (90 %) with much smaller amounts of
N-(2-methylbutyl)propanamide and heptacosane. Perkins et al. (1990a) examined an
acetate extract of rectal glands of sexually mature male B. dorsalis from a colony
maintained in Hawaii and detected the trimethyl ester of citric acid (a major compo-
nent), the trimethyl ester of phosphoric acid, and dimethyl succinate along with
methyl esters of fatty acids and two spiroacetals. The males sampled in this study
had not fed on ME, and no biological activity was demonstrated for the compounds
identified. However, Nishida et al. (1988a, b) and Tan and Nishida (1996) demon-
strated that males of B. dorsalis and B. papayae transformed consumed synthetic ME
to two major pheromonal components – E-coniferyl alcohol (ECF) and 2-allyl-4,5-
dimethoxy phenol (DMP), with trace quantity of Z-3.4-dimethoxycinnamyl alcohol
(detected in some males). Nishida et al. (1988a) also detected these compounds in
wild B. papayae males, indicating the males had fed on ME-bearing plants in the
field, and a later study (Tan et al. 2002) showed that B. papayae males that fed on an
ME-bearing orchid flower contained ECF and DMP in the rectal gland (Fig. 2.2). In
laboratory tests, males deprived of ME did not have ECF or DMP in the rectal gland.
As an aside, B. papayae males visiting an orchid whose floral fragrance contained
zingerone (a compound structurally similar to ME) were found to have zingerol in the
rectal gland, suggesting a role in pheromone synthesis for this compound as well (Tan
and Nishida 2000, 2007). More recently, Tan et al. (2011, 2013) found ECF and DMP
in the rectal sac of ME-fed males of B. invadens and B. philippinensis. Males of
B. carambolae differ from the aforementioned species in that they produce only ECF
after ingesting ME (Tan and Nishida 1996; Wee and Tan 2005a). Moreover, the sex
pheromone of B. carambolae contains larger amounts of endogenously produced
compounds, including 6-oxo-1-nonanol (a major component that is also detected in a
closely related sibling species, Bactrocera occipitalis (Bezzi) and a distant species,
B. umbrosa (Perkins et al. 1990b)) and three minor components, N-3-methylbutyl
acetamide, ethyl benzoate, and 1,6-nonanediol (Wee and Tan 2005a).
Since Nishida et al.’s reporting, a number of studies have demonstrated that ME
consumption increases male mating success in several species in the B. dorsalis
complex (Shelly and Dewire 1994; Shelly et al. 1996; Shelly 2010a; Tan and
Nishida 1996, 1998; Wee et al. 2007; Orankanok et al. 2013; Obra and Resilva
2013). However, the role of pheromone composition in determining this outcome is
not known with certainty. In laboratory cage assays, Kobayashi et al. (1978) dem-
onstrated attraction of B. dorsalis females to both live males and male rectal gland
extract even when males were not previously fed ME. Wee and Tan (2005a)
likewise reported zigzag anemotaxis by B.carambolae females to live males and
endogenously produced rectal gland substances. Thus, the breakdown products of
ME are not necessary to elicit female response. Nonetheless, using a wind tunnel or
laboratory cages, several studies on B. dorsalis complex species (Shelly and Dewire
1994; Hee and Tan 1998; Wee et al. 2007) have reported greater female attraction to
males that had previously fed on ME than to unfed males, and Hee and Tan (1998)
and Khoo et al. (2000) showed female attraction to ECF and DMP individually
(with greater attraction to ECF than DMP in these tests) and in combination
(Fig. 2.3). Importantly, greater female response to ME-fed males has been
26 K.H. Tan et al.

documented, not only using synthetic ME, but also after male feeding on natural
floral (Shelly 2000a, 2001a) or fruit (Shelly and Edu 2007) sources of ME. Several
studies (Hee and Tan 1998; Wee and Tan 2005a; Wee et al. 2007) have documented
maximum female attraction to male sex pheromone at dusk, the time of peak sexual
activity in B. dorsalis species complex.
To our knowledge, only two studies have examined the long-range attractiveness
of male pheromone to females in the field. In a study examining female attraction to
groups (leks) of varying size, Shelly (2001b) placed B. dorsalis males (none of
which had fed on ME) in screen-covered cups, which were in turn placed on trees
situated in a circular (10 m radius) array around a central female release point.
Approximately 10 % of released females were sighted near male-containing cups
over all groups. In a second study also conducted on B. dorsalis in Hawaii, Shelly
(2001c) performed two experiments in which groups of (i) ME-fed or ME-deprived
males or (ii) flower-fed or flower deprived males (where the flower used
Fig. 2.2 Acquisition and biotransformation of methyl eugenol to sex pheromone by Bactrocera
dorsalis males
Fig. 2.3 Bactrocera dorsalis females and males attracted to E-coniferyl alcohol. A An attracted
female with ovipositor extruded (arrow), B Aggregation of males and an attracted female with
extruded ovipositor at bottom left

[puakenikeni, Fagraea berteriana A. Gray ex Benth] was known to contain
ME-like compounds [Nishida et al. 1997]) were placed in cups suspended in host
trees (one male type [i.e., fed or non-fed] per tree) situated in a circle (12 m radius),
and females were released from the center. Compared to non-fed males, both ME-
and flower-fed males were found to signal more frequently and attract greater total
numbers of females as well as greater numbers of females per signaling male. These
studies were not designed to test explicitly the function of pheromone signaling
(since blank controls were not run in either study), but they nevertheless hint at
long-range attraction mediated by male pheromone and thus suggest the potential
for male pheromone as a trap bait for species in the B. dorsalis complex.
Data on pheromonally-mediated male-male attraction are inconsistent. In labo-
ratory cages, B. dorsalis males showed no attraction to conspecific males (non-ME-
fed, Kobayashi et al. 1978). In contrast, Nishida et al. (1988b) found that traps
baited with DMP captured as many wild males as traps baited with ME. In wind
tunnel tests, Hee and Tan (1998) found that B. papayae males were attracted to both
ME-fed and control (unfed) conspecific males but showed greater attraction to the
treated males. Also using a wind tunnel, Wee et al. (2007) found non-ME-fed males
of B. carambolae were attracted to ME-fed conspecific males at a much higher level
than observed in the converse situation (i.e., ME-fed males responding to non-ME-
fed males). Moreover, field cage observations showed that unfed males aggregated
around ME-fed males and fed on anal secretions of ME-fed males (see also Tan and
Nishida 1996). Results for B. papayae and B. carmabolae thus suggest that male
sex pheromone may also serve as an aggregation pheromone. However, this
function implies an evolutionary advantage to aggregation per se (e.g., increased
mating success), whereas the possibility remains that male-male attraction simply
represents a special case of male attraction to ME (or ME-like compounds), where
the ME source is a male rather than a plant.
2.2.2 Presumed Sex Pheromone of Two Sibling Species of B. zonata
Complex
ME also acts as a pheromone precursor for both B. correcta and B. zonata. In
B. zonata, it is transformed to two male sex pheromonal components, DMP and Z-
coniferyl alcohol (ZCF), although final confirmation awaits tests of biological
activity on female response (Tan et al. 2011). In B. correcta, however, ME is
converted to ZCF and (Z)-3,4-dimethoxycinnamyl alcohol (ZDMC) (Tokushima
et al. 2010). Furthermore, wild B. correcta males also accumulate large quantities
of sesquiterpene hydrocarbons, namely β-caryophyllene, α-humulene, and
alloaromadendrene, in the rectal gland in addition to, or instead of, ZCF and
ZDMC (Tokushima et al. 2010). The distinct difference in sex pheromonal profiles,
albeit having a common ZCF component, between the two sibling species, most
likely, plays an important role in maintaining reproductive isolation.
Interestingly, recent comparative field tests conducted in Thailand during 2012–
2013 and based on average flies/trap/day using a similar lure dosage per trap
28 K.H. Tan et al.

showed that β-caryophyllene caught on average 7 (range 3–16) times more
B. correcta wild males than ME during the first 3 days of trapping (Tan,
Chinvinijkul, Wee Nishida, unpublished data). This is the first case of a lure
being more attractive than the very potent ME to a ME-sensitive Bactrocera
species. Therefore, further behavioral/ecological studies, especially related to the
role of the sequiterpene and its possible replacement of ME in the trapping of
B. correcta, are warranted.
2.2.3 Sex Peromone of B. umbrosa
Rectal gland extracts showed the presence of (E)- and (Z)-2-methyl-1,6-dioxaspiro
[4.5]decanes, 3-methylbutanol, 1,7-dioxaspiro [5.5]undecane, and 6-oxononan-ol
(Perkins et al. 1990b). In addition, some unidentified ME metabolites (identities
currently being evaluated) were detected in the rectal gland after consumption of
ME by males (Nishida and Tan, unpublished data). In Malaysia, B. umbrosa and
B. papayae are endemic and sympatric species as well as serious pests of jackfruit,
Artocarpus heterophyllus Lam., but they do not interbreed. Apparent reproductive
isolation was observed between the two species even when both males and females of
both the species were kept together in a cage for approximately 2 months; intraspe-
cific but no interspecific matings were observed (Tan, unpublished observations).
2.2.4 Sex Pheromone of B. cucurbitae and B. tryoni
Males of these species are attracted to RK/CL. Rectal gland secretions of
B. cucurbitae contain N-3-methylbutyl acetamide, two spiroacetals, and three
pyrazines (Baker et al. 1982a; Baker and Bacon 1985). Later, ethyl
4-hydroxybenzoate (a major component) and propyl 4-hydroxybenzoate (a minor
component) were also detected in the rectal gland of the melon fly (Perkins
et al. 1990b). Nishida et al. (1990) showed that sexually mature male melon flies
produce, endogenously in the rectal gland, relatively small quantities of N-3-
methylbutyl acetamide, methoxy-acetamide, methyl, ethyl, and propyl
4-hydroxybenzoate, and a large quantity of 1,3-nonanediol, which was not detected
in the previous studies. The amounts of 1,3-nonanediol and ethyl 4-hydroxybenzoate
stored in the rectal gland increased with age, starting 2 weeks after adult eclosion,
thus coinciding with attainment of sexual maturity (Nishida et al. 1990). Addition-
ally, at sexual maturity males of B. cucurbitae consume and sequester RK from
anthropogenic (Nishida et al. 1990) and natural (Nishida et al. 1993; Tan and Nishida
2005) sources into the rectal gland. As noted above for B. papayae, males of
B. cucurbitae are also attracted to and feed on zingerone, an orchid floral volatile,
and store it unmodified in the rectal gland (Tan and Nishida 2000).
Males of B. tryoni produce endogenously six amides as major sex pheromonal
components, and three of the six, namely, N-3-methylbutyl acetamide (MBA), N-3-
methylbutyl propanamide (MBP), and N-3-methylbutyl-2-methyylpropanamide,

are frequently detected in the rectal gland (Bellas and Fletcher 1979). Furthermore,
MBA and MBP increase significantly from 14 to 17 day-old males corresponding
with attaining sexual maturity (Tan and Nishida 1995). This suggests that the two
chemicals may act as close range sex pheromone. Males consume plant-borne RK
or RK from spontaneous hydrolysis of CL and sequester it in the rectal gland as a
major pheromonal component (Tan and Nishida 1995).
Analogous to the B. dorsalis complex, ingestion of CL/RK has been shown to
enhance male mating success, though the effect appears short-lasting both for
B. cucurbitae (1 day after feeding, Shelly and Villalobos 1995; Shelly 2000b) and
B. tryoni (1–3 days after feeding, Kumaran et al. 2013). More recently, B. tryoni
males fed zingerone were also found to have a mating advantage over control males
deprived this compound (Kumaran et al. 2013). The role of the sex pheromone in
influencing male mating success is unknown. Kobayashi et al. (1978) found that
B. cucurbitae females were attracted to male rectal glands as well as live males
(in neither case were males fed CL/RK) but that the attraction was far weaker than
that observed for B. dorsalis females to conspecific males. In wind tunnel trials,
Khoo and Tan (2000) demonstrated that CL-fed and zingerone-fed males of
B. cucurbitae attracted more females compared to males deprived these com-
pounds, which strongly suggests a sex pheromonal role for these exogenous
phenylbutanoids. To our knowledge, there are no laboratory or field data available
investigating the effect of the male sex pheromone on female attraction or male
mating success in B. tryoni.
2.2.5 Sex Pheromone of Bactrocera oleae
Bactrocera oleae (Rossi) [formerly Dacus oleae (Gmelin)], the olive fruit fly,
unlike the other major pest Bactrocera species mentioned above, is a monophagous
pest species. Additionally, the species differs from other Bactrocera in that the
B. oleae females attract males for mating and not vice versa (Haniotakis 1974; but
see below). Baker et al. (1980) identified the major component of the female sex
pheromone as (1,7-dioxaspiro[5.5]undecane (also known as olean; as noted above,
this compound was also identified from the pheromone of B. umbrosa). Additional
studies (Mazomenos and Haniotakis 1981) confirmed this finding and also identi-
fied three minor components, o-pinene, n-nonanal, and ethyl dodecanoate, in the
female pheromone (see also Baker et al. 1982b, who identified two hydroxyspir-
oacetals from B. oleae females). Other components of the female sex pheromone
were reported (Gariboldi et al. 1982), but their isolation and biological activity
(tested with synthetic products) was not corroborated (Jones et al. 1983;
Mazomenos 1989). Interestingly, olean was also isolated from the rectal gland of
male B. oleae along with other components (Mazomenos and Pomonis 1983).
Canale et al. (2012) reported that, among males, olean production is greatest
among young males (5–8 days old) and then ceases by 11 day of age. Also, in a
recent finding, Carpita et al. (2012) identified (Z)-9-tricosene from male rectal
gland extracts and reported female attraction to this compound in synthetic form.
30 K.H. Tan et al.

Several studies (Haniotakis 1974; Mazomenos and Haniotakis 1981, 1985) have
demonstrated male attraction to natural or synthetic components or whole blends of
the female pheromone in B. oleae. Laboratory and field experiments demonstrated
that olean was more attractive than the remaining three components but that the
combination of all four components was more attractive than olean alone. More
detailed chemical analysis (Haniotakis et al. 1986a) revealed that olean exists as (R)
and (S) mirror (stereo) image enantiomers, (R)-olean and (S)-olean (Fig. 2.4) and
that (i) males are more strongly attracted to (R)-( )-oleanthan(S)-(+)-olean, (ii) the
converse was true for females, and (iii) overall, males showed greater attraction to
response to the compound than did females. Haniotakis et al. (1986a) suggest olean
may serve an aggregation or aphrodisiac function for females. Relative to the strong
evidence gathered for male attraction to the female sex pheromone, data regarding
female attraction to male olfactory signals are less conclusive. Mazomenos and
Pomonis (1983) reported negligible female response in laboratory tests to extracts
of rectal glands of mature males. More recently, however, Mavraganis et al. (2010)
demonstrated that whole body extracts of B. oleae males were highly attractive to
females and suggest that the previous negative results may have reflected low
pheromone concentrations in the rectal gland extracts compared to those of whole
body. Benelli et al. (2013) found that young males, which, as noted above, produce
olean at higher levels than old males, did not have a mating advantage over older
individuals.
In contrast to the other economically important species discussed here, several
studies have demonstrated the usefulness of olean in baiting traps. In general,
because olean is primarily a male attractant, the most effective traps appear to be
those that combine the pheromone with ammonium or some other food bait that
targets females (Haniotakis and Vassiliou-Waite 1987; Broumas and Haniotakis
1994). Traps baited with this combination have been used both in detection (Rice
et al. 2003; Yokoyama et al. 2006) and in mass-trapping efforts to lower olive
infestation (Haniotakis et al. 1986b, 1991; Iannotta et al. 1994; Petacchi et al. 2003;
Noce et al. 2009; see also Navarro-Llopis and Vacas, Chap. 15, this volume).
(R)-Olean (S)-Olean
[(R)-1,7-dioxaspiro[5.5]undecane] [(S)-1,7-dioxaspiro[5.5]undecane]
O O O O
Fig. 2.4 Stereo enantiomers of (R)- and (S)-olean found in B. oleae sex pheromone

2.2.6 Synthesis
Because males of many economically important Bactrocera species are strongly
attracted to male lures (see below), the development of pheromone-based trapping
for this genus would target females primarily. The important finding that ME and
CL/RK are used in pheromone synthesis and that their incorporation in this process
enhances the attractiveness of the male olfactory signal could facilitate the produc-
tion of effective pheromone baits. Nonetheless, many of the obstacles noted above
for Anastrepha apply to Bactrocera as well, namely the lack of data on (i) the long-
range attractiveness of the male pheromone, (ii) the optimal blend (relative
amounts) and release rate of pheromonal components that produce maximal attrac-
tiveness, and (iii) the importance of olfactory signals relative to other modalities
(i.e., visual, acoustic) in the mating behavior of Bactrocera species
2.3 Ceratitis Pheromones
The genus Ceratitis contains approximately 80 species, most of which are found in
tropical Africa, although C. rosa Karsch (Natal fruit fly) has invaded Mauritius and
Réunion and C. capitata (Wiedemann) (Mediterranean fruit fly or medfly) has
spread globally (South and Central America, Western Australia, and Hawaii)
(De Meyer 2000). The medfly is, of course, one of the most harmful agricultural
pests worldwide, with females ovipositing in soft fruits of more than 300 plant
species (Liquido et al. 1990). Other major economic pests in the genus include
C. rosa, C. cosyra (Walker) – mango fruit fly, and C. catoirii Guérin-Méneville
(White and Elson-Harris 1992). Because of its economic importance, the medfly
has been studied far more intensively than its congeners, and this review will
necessarily focus on this species.
Féron (1959, 1962) provided the first detailed description of calling behavior in
C. capitata males, which he associated with the emission of volatiles attractive to
females. While the notion of male-produced olfactory stimuli had been proposed
decades earlier (Martelli 1910; Back and Pemberton 1918, both cited in Jones
1989), Féron supplied empirical evidence by reporting female attraction to a cotton
wick previously exposed to calling males. Quilici et al. (2002) and Briceño
et al. (2005) report similar pheromone-calling behavior in C. rosa and C. catoirii,
but data showing female attraction to calling males are not yet available for these
species. For the medfly, Ohinata et al. (1977) and Nakagawa et al. (1981a) provided
the first quantitative demonstration of the long-range, female attraction to calling
male in the field by recording female captures in male-baited traps. Female attrac-
tion to live, calling medfly males (or their odor) was reported in further laboratory
(Landolt et al. 1992a; Jang 1995; Jang and Light 1996; Jang et al. 1994, 1998) and
field (Shelly 2000c) studies. The importance of olfaction to females has been
demonstrated conclusively via antennal ablation: in existing studies, females with
32 K.H. Tan et al.

antennae removed either mated not at all (Nakagawa et al. 1973; Levinson
et al. 1987) or at very low levels (Shelly et al. 2007).
Research aimed at characterizing the chemical composition of the male sex
pheromone in C. capitata and identifying the biologically active components was
undertaken even before female attraction to calling males was demonstrated in the
field. Jacobson et al. (1973) identified two putative pheromonal components –
methyl (E)-6-nonenoate and (E)-6-nonen-1-ol – and indicated that females were
attracted to both compounds in assays performed in small cages. Ohinata
et al. (1977, 1979) identified the same two components as well as 15 carboxylic
acids, which were presumed to ‘activate’ the two main components. However,
contrary to Jacobson et al. (1973), various blends of these different chemicals were
found to attract males but not females. Delrio and Ortu (1987, cited in Millar 1995)
likewise reported no female attraction to methyl (E)-6-nonenoate. Jacobson and
Ohinata (1980) also reported ( )-β-fenchol in the medfly sex pheromone, but
subsequent analyses failed to detect this compound.
In fact, it appears that, due to inadequate analytical methods, initial efforts to
identify pheromonal components led to inaccurate results, which could not be
confirmed by later studies. Despite the potential usefulness of pheromone-based
lures in medfly surveillance programs, relatively few studies have further investi-
gated pheromonal composition and/or the role of particular components as female
attractants. Baker et al. (1985) identified nine components in male medfly emis-
sions, with the three most abundant being ethyl (E)-3-octenoate, geranyl acetate,
and (E, E)-α-farnesene. They further proposed that another component,
3,4-dihydro-2-H-pyrrole (1-pyrroline), functioned as the key attractive element to
females (although no data on its purported biological activity were provided). In a
follow-up study, Baker et al. (1990) tested the attractiveness of four compounds
(linalool, two pyrazines, and geranyl acetate) in field trials in Mexico. Both
individually and in various blends, these chemicals attracted both sexes of
C. capitata, although the olfactory stimuli used bore little resemblance to the
emissions of calling males.
More thorough chemical analyses (Jang et al. 1989a; Flath et al. 1993) confirmed
the presence of the nine components reported by Baker et al. (1985), with one
exception, and revealed a pheromonal complexity far greater than previously
documented. Jang et al. (1989a) identified a total of 56 compounds from the odor
of calling males, and Flath et al. (1993) identified four additional compounds, thus
revealing that the sex pheromone of C. capitata males consists of approximately
60 different compounds. Jang et al. (1989b) established four abundance categories
for the pheromonal constituents, with five considered major components (ethyl
acetate, 1-pyrroline, ethyl (E)-3-octenoate, geranyl acetate, and (E,E)-α-farnesene).
Based on electroantennogram (EAG) recordings, ethyl acetate, 1-pyrroline, and (E,
E)-α-farnesene elicited low EAG responses (relative to a standard), geranyl acetate
elicited a moderate response, and ethyl (E)-3-octenoate elicited a high response.
Overall, the sexes displayed similar EAG responses to the different pheromonal
components. Additional studies (Casaña-Giner and Primo-Millo 1999; Gonçalves
et al. 2006) have identified additional components from the volatiles of calling

C. capitata males, and Cossé et al. (1995) confirmed strong female EAG responses
to some of the previously identified compounds of the male pheromone.
Since Jang et al.’s (1989b) seminal paper, few studies have examined behavioral
responses to the sex pheromone of C. capitata males. These investigations have
employed simplified blends, including only the major components or a subset
thereof, owing to (i) the difficulty of creating close mimics to the naturally complex
male odor and (ii) the assumption that only a small portion of the pheromonal
chemicals identified actually have biological activity (Landolt et al. 1992a). Thus,
working in a coffee field in Guatemala, Heath et al. (1991) demonstrated attraction
of medfly females to a synthetic blend containing three of the major components
(ethyl (E)-3-octenoate, geranyl acetate, and (E, E)-α-farnesene). In two testing
periods (lasting 6 and 8 days, respectively), 259 and 368 wild females, respectively,
were captured in traps baited with the synthetic blend. However, the effectiveness
of the blend as a trap-bait, could not be ascertained, because (i) no estimates of the
size of the wild population were made and (ii) no traps baited with live males were
operated, thus precluding assessment of the relative competitiveness of the simpli-
fied blend. Working with the same 3-component blend, Landolt et al. (1992a)
reported an oriented response (i.e., upwind movement coupled with course-
correcting, zigzagging flight) of female medflies to the stimulus in a wind tunnel.
However, only a small proportion (3 %) of the females actually contacted the odor
source, a level not significantly different from the contact rate observed in the
absence of an olfactory stimulus. Jang et al. (1994) studied female response to live
males, each of the five major components, and a mixture of these five compounds in
a wind tunnel. Although females showed greatest attraction to live males, the five
component blend was more attractive than the individual compounds and appeared
to elicit a much greater female response than the 3-component blend used by
Landolt et al. (1992a). While the above studies reveal the attractiveness of simple
pheromone blends, Casaña-Giner et al. (2001) reported very low catch of female
medflies in traps baited with a 6-component mixture and questioned the long-range
effectiveness of male pheromone-baited traps.
Adopting a different approach, Mavraganis et al. (2008) obtained whole body
extracts of medfly males and monitored female attraction to complete extracts of
laboratory vs. wild males as well as the major components of the extracts either
individually or in different combinations. Interestingly, females showed greater
attraction in laboratory assays to the extracts from wild males than laboratory
males. Samples from wild males contained larger amounts of the compound
α-copaene than those from laboratory males, and this compound was found to
have greatest attractancy to females in comparisons of the individual components.
Field trials further revealed that total male extracts as well as synthetic blends of
major components were highly attractive to wild females, the majority of which
were virgin. Thus, in contrast to other studies (Heath et al. 1991; Casaña-Giner
et al. 2001), the total extracts and blends tested by Mavraganis et al. (2008) appear
to be highly attractive to female medflies and clearly merit additional field testing.
The chemical complexity of the male sex pheromone has, it appears, discour-
aged efforts to develop or improve the attractiveness of synthetic sexual lures to
34 K.H. Tan et al.

medfly females. Not only might some trace components await identification, but
knowledge of the relative amounts of the constituent compounds is imprecise. In
addition, the blend containing the five major components was far less attractive to
females than the odor emitted by live males, suggesting that simplified formulations
will not be able to compete against calling males in the wild (Jang et al. 1994).
Similarly, whole body extracts were far more attractive to wild females than
simplified blends with relatively few components (Mavraganis et al. 2008). In
this regard, Howse and Knapp (1996), noting similarity in the volatiles released
by host fruit, citrus in particular, and calling male medflies, suggest that competi-
tion with host fruit odors may further limit the effectiveness of male pheromonal
traps in orchards (but see Mavraganis et al. 2008). In addition to the chemical
composition, the importance of release rate in the development of a potent
pheromone-based lure is uncertain as relevant data are inconsistent. In particular,
results from Ohinata et al. (1977) and Jang et al. (1994) suggest that the amount of
male emission does not have a marked effect on its attractiveness, whereas Heath
et al. (1991) found that an intermediate release rate resulted in higher female
captures than lower or higher rates. Finally, while the identification of a female
lure for Ceratitis species is recognized as a worthy research objective, the wide
usage of a male-lure (trimedlure) may lessen the impetus to achieve this goal.
2.4 Dacus Pheromones
To date, little effort has been devoted to identifying possible sex or aggregation
pheromones of Dacus species. This lack of interest is probably due to a combination
of several factors, such as insufficient funding, the small number of pest species in
the genus, which are generally moderate pests relative to highly invasive
Bactrocera species, and the availability of male lures for surveillance purposes.
2.5 Rhagoletis Pheromones
Male sex pheromones have been demonstrated in several Rhagoletis species. Using
caged host trees, Prokopy (1975) and Katsoyannos (1976) furnished evidence for a
male sex pheromone in R. pomonella (Walsh), the apple maggot fly, and R. cerasi
Loew, the cherry fruit fly, respectively, by reporting attraction of mature, virgin
females to cages of live males as well as to empty cages that had housed males. No
male-to-female or male-to-male attraction was observed in either of these species.
Also, males of these two species did not display any behavior typically associated
with pheromone release in other tephritids (e.g., wing fanning), and consequently
the manner of pheromone release was unclear. Additional tests on R. cerasi further
showed that immature and mated females do not respond to male pheromone and
that mature virgin females responded to an extract obtained from whole body

preparations of mature males (Katsoyannos 1982). In addition to the male phero-
mone, several studies (Prokopy and Bush 1972; Katsoyannos 1975) have shown
that female host marking pheromone acts as a male arrestant, possibly to increase
the frequency of intersexual encounters. Research on sexually-oriented, phero-
monal communication has not continued beyond these few studies, and the poten-
tial use of male pheromones in Rhagoletis trapping or detection has not been
investigated.
2.6 Toxotrypana curvicauda Pheromone
Landolt and Hendrichs (1983) reported “puffing” of the pleural areas of abdomen in
male Toxotrypana curvicauda Gerstaecker, the papaya fruit fly, a behavior associ-
ated with pheromome release in other tephritids, and Landolt et al. (1985) later
demonstrated female attraction to male pheromone in laboratory assays, including
wind tunnel trials. The pheromone has a single chemical component, which was
identified as 2-methyl-6-vinylpyrazine (Chuman et al. 1987). Additional observa-
tions (Landolt and Heath 1988; Landolt et al. 1991) showed that the pheromone
attracts, not only virgin females, but also mated females and males and that female
response is increased when green papaya fruit or an extract thereof was presented
with the male pheromone (Landolt et al. 1992b).
The male pheromone with sticky-coated green spheres was tested in Florida and
resulted in high captures of T. curvicauda females (Landolt et al. 1988; Landolt and
Heath 1990). To facilitate field use, Heath et al. (1996) developed a membrane-
based formulation system and showed that release rates, which were dependent on
the amount of pheromone loaded into the system, were relatively constant over
trials lasting 23 days. They also showed that green opaque cylindrical traps yielded
higher captures than spherical traps. In field tests conducted in a papaya orchard in
Guatemala, greater numbers of females were, as expected, captured in pheromone-
baited than blank cylinders. Surprisingly, however, similar tests run in a Mexican
papaya plantation detected no influence of pheromone presence/absence on female
captures in green cylindrical traps. Reasons for this discrepancy are unknown, and
additional field trials are required to evaluate the efficacy of pheromone-baited traps
in detecting and/or suppressing populations of T. curvicauda.
3 Male Lures
There are two types of male lures: anthropogenic (e.g., CL, trimedlure [TML],
fluorinated methyl eugenol analogs, raspberry ketone-formate (RKF)) and plant-
borne (e.g., α-copaene, ME, RK, and zingerone). For certain species, male lures are
relatively cheap to synthesize due to the simplicity of the chemical structures,
which are often not stereoisomeric. In addition, they are very potent attractants in
36 K.H. Tan et al.

most cases and thus appear a more robust option for trapping programs than the
development of the male sex pheromone as baits. As a result, they are frequently
used as baits in trapping for surveys and detection of invasive species, delimitation
of an infestation, and control or eradication via the male annihilation technique.
Because of their importance in trapping, numerous reviews (Chambers 1977;
Cunningham 1989; Metcalf 1990; Millar 1995; Jang and Light 1996; Sivinski and
Calkins 1986; Oliver et al. 2004; Vargas et al. 2010a) of male lures already exist,
and rather than re-hashing information, our present aim was to address a few
selected topics.
3.1 Anastrepha
At present, there are no identified male lures for any Anastrepha species. This fact
does not reflect a lack of effort. Approximately 8,000 compounds were screened as
possible attractants for A. ludens (Chambers 1977) and 1,320 compounds were
tested for A. suspensa (Burditt and McGovern 1979; cited in Cunningham 1989).
Despite intense screening, no male attractant was identified in both the
investigations.
3.2 Bactrocera
While most species in the genus remain untested, Bactrocera has been broadly
categorized into three groups of species based on male response to two very potent
attractants (Drew 1974; Hardy 1979; Drew and Hooper 1981; White and Elson-
Harris 1992; Fig. 2.5). Nearly 200 species have been identified as CL/RK
responders and 81 species as ME-responders (IAEA 2003). A third group includes
approximately 15 species (limited to an Australian survey) that do not respond to
either CL/RK or ME as evidenced by their absence in traps from areas where
species were known to be present (Drew and Hooper 1981). No species has been
identified that responds to both CL/RK and ME. Response to these compounds
correlates with morphologically-based taxonomic classification (Drew 1974; Drew
and Hooper 1981), and several authors (Metcalf et al. 1979, 1981, 1983, 1986;
Metcalf 1990; Raghu 2004) suggest that the existence of distinct CL/RK- and
ME-responding species groups reflects evolutionary divergence from a common
saprophytic relationship with rotting fruits. Broadly, coevolution between plants,
specifically the appearance of novel metabolic pathways and the subsequent inte-
gration of those products into essential oils, and dacine tephritids, specifically
adaptation of olfactory receptors to chemically diversified plant essential oils, is
considered to underlie the present-day CL/RK or ME distinction in species
responsiveness.

3.2.1 Methyl Eugenol
ME is widely recognized as the most powerful male lure currently in use for
detection, control, and eradication of any pestiferous tephritid species. The chemical
occurs naturally in more than 450 plant species representing 80 families spanning
38 different orders in varying amounts, from a trace quantity to over 90 %, in
essential oils extracted from flowers, leaves, roots, stems, or whole plant extracts
(see review by Tan and Nishida 2012). It was first discovered as a fruit fly attractant
by Howlett (1912, 1915), who observed males of B. dorsalis and B. zonata
responding to ME-containing citronella grass, Cymbopogon nardus (L.) Rendle.
Steiner (1952) further documented the strong attraction of B. dorsalis male to ME
and noted their vigorous feeding on the chemical. Metcalf et al. (1975) exposed
B. dorsalis males to ME as well as 34 analogs and found that ME elicited the greatest
feeding response. The powerful attraction of this lure was further demonstrated by
(a) a simple test in which approximately 1 nanogram (10 9
g) of ME spotted on a
silica gel TLC plate placed in the field attracted B. papayae males, which readily
consumed the minute amount of attractant (Tan and Nishida 2000); and (b) trap
placement for trapping B. dorsalis and B. umbrosa hung at different heights – ground
level (0.3–0.5 m), below (1.5–2 m), middle (5–7 m) and above (10–12 m) tree canopy
– using traps baited with 0.5 ml ME/trap and set up using a 4 4 Latin square design
in a 5 ha Penang village (a 4-day experiment conducted in two fruiting seasons),
showed no significant difference in daily fly captured (flies/trap/day) (Tan 1984).
Because of its potency, ME-baited traps have been used in a variety of ways
including (i) detection and surveillance of invasive species (Drew et al. 2005;
McQuate et al. 2008a; Jessup et al. 2007), (ii) quarantine surveys and delimitation
RO
O
Latilure(a-ionol)
OH
Bactrocera male attractants
Methyl eugenol
CH3O
CH3O
Zingerone
HO
O
CH3O
R = H : Raspberry ketone
R = CH3CO : Cue-lure
R = OHC : Raspberry ketone-formate
3-Oxo-7,8-dihydro-a-ionone
O
O
CH3O
CH3O
OR
R = H : (E)-3,4-Dimethoxycinnamyl alcohol
R = COCH3 : (E)-3,4-Dimethoxycinnamyl acetate
Phenylpropanoids Phenylbutanoids a-Ionone analogs
Fig. 2.5 Chemical structures of lures for Bactrocera males
38 K.H. Tan et al.

(Allwood 2000; Sookar et al. 2008; OEPP/EPPO 2010), (iii) suppression and
eradication (Steiner et al. 1965; Hancock et al. 2000; Hsu et al. 2010; Vargas
et al. 2008), and (iv) ecological studies, including faunal surveys (Tan and Lee
1982), population dynamics and phenology (Tan 1985; Tan and Serit 1988, 1994;
Ye and Liu 2005; Han et al. 2011; Kamala Jayanthi and Verghese 2011), adult
survivorship (Tan and Jaal 1986), and dispersal (Iwahashi 1972; Tan and Serit
1988; Chen et al. 2006; Froerer et al. 2010). As noted above, existing reviews
address many of these topics (see, in particular, Vargas et al. 2010a, b), and here we
briefly address three topics, namely (i) the effect of ME feeding on subsequent
attraction to ME-baited traps , (ii) age-dependent variation in response to ME, and
(iii) interspecific differences in attraction to ME.
There is some evidence that feeding on ME reduces the likelihood of future ME
feeding. Males of B. dorsalis given access to ME for 0.5–24 h prior to release were
captured in ME-baited traps at much lower rates (1–4 %) than control, unfed males
(22 %; Shelly 1994). In a second test, B. dorsalis males exposed to ME for 2 h and
then held for 7–35 days prior to release were also captured at lower rates (11–18 %)
than control, unfed males (34 %). Other studies, however, indicate that these results
may be an artifact of the experimental design. When treated males were provided
ME-bearing flowers instead of commercial ME, there was no difference in capture
rate in ME-baited traps between floral-exposed and control males, indicating that
the unnaturally high purity and availability of synthetic ME in the previous study
may have accounted for the diminished capture rate of treated males (Shelly
2000d). Moreover, dissection of wild males of several Bactrocera species attracted
to ME-baited traps (designed to prevent feeding) revealed the presence of ME
metabolites in the rectal gland of nearly all individuals. Wee and Tan (2001)
extracted the rectal gland from 76 wild B. papayae males in Penang, Malaysia,
and found that all individuals possessed some ME-metabolites, ranging from trace
quantities to approximately 103 μg per male. Similarly, Tan et al. (2011) reported
that nearly all wild-caught B. invadens and B. zonata males dissected contained at
least trace amounts of ME metabolites (maximum observed: 10 μg per gland for
both species). Interestingly, after ad libitum feeding on synthetic ME, laboratory
measurements regarding accumulation of ME breakdown products showed that
males of B. papayae, B. invadens, and B. zonata sequester, on average, 20, 170,
and 25 μg/gland 1–2 days after feeding (Wee and Tan 2001; Tan et al. 2011; see
also Tokushima et al. 2010 for comparable data on B. correcta). As the quantity of
ME derivatives detected in wild males attracted to ME traps was often less than
these averages, it appears that, in general, males are unable to “tank up” at any one
ME source and therefore must visit multiple ME sources to gather a sufficient
amount of the chemical, a result consistent with the aforementioned result regard-
ing the high capture rate at ME traps of B. dorsalis males experimentally fed
ME-bearing flowers in the laboratory (Shelly 2000d).
Interestingly, when B. papayae males were exposed to commercial ME (isolated
to prevent feeding) for various time periods within a trap, they became habituated
after an hour and would not respond to subsequent ME exposure for a week
(unpublished data, discussed in Tan et al. 2002). Because of this, the trapped

males were removed from ME-traps every 0.5 h to avoid possible habituation to ME
when using the mark-release-recapture technique to estimate population size (Tan
1985). While access to commercial ME of high purity may induce habituation
depending on the length of exposure and underestimate the incidence of repeat ME
feeding in nature, it nonetheless suggests a means of improving the effectiveness of
control programs against B. dorsalis (or related species), namely the simultaneous
application of the male annihilation and sterile insect techniques. Providing sterile
males access to ME before release may both increase their mating competitiveness
(McInnis et al. 2011) and reduce their attraction to insecticidal-laden ME sources
deployed for male annihilation. Barclay and Hendrichs (Chap. 11, this volume)
examine the improved control afforded by this strategy through a modeling
approach.
As another caveat, to the extent that male responsiveness to ME has a heritable
component, it seems possible that prolonged application of a male annihilation
program might select for males showing low attraction to ME, thus eclipsing the
effectiveness of the program. Faced with persistent populations of B. dorsalis on
several Japanese Islands despite prolonged attempts at male annihilation, Itô and
Iwashashi (1974) and Habu et al. (1980, 1984) suggested that selection for
ME-insensitive males was responsible, and, as a result, SIT was implemented and
finally achieved eradication. In support of their claim, Itô and Iwahasi (1974)
exposed B. dorsalis males to ME in the laboratory and selected non-responding
males as sires. Within only two generations of such selection, they produced a strain
with lower ME responsiveness than a control line. Working with B. dorsalis in
Hawaii, Shelly (1997) likewise reported a consistent reduction in ME responsive-
ness over 8 generations for several lines sired by non-responding males. These
studies indicate that, while the evolution of lure-insensitivity has not been demon-
strated conclusively, programs of male annihilation are most effective when applied
intensely with the aim of rapid eradication.
Age-dependent response to ME has been examined in some detail for
B. dorsalis. While all studies confirm that ME response increases with male age,
they differ in their estimates of ME responsiveness among immature individuals.
On one hand, several studies (Umeya et al. 1973; Itô and Iwahashi 1974; Habu
et al. 1980; Tan et al. 1987) report no or very little attraction by very young males
(1–5 days-old) and a close association between ME response and male sexual
maturation (see also Fitt 1981b for data on B. opiliae). Tan et al. (1987), for
example, found that less than 2 % of 5 days-old laboratory-reared males responded
to ME in a wind tunnel and no wild males, emerged from naturally infested star
fruits (Averrhoa carambola Linn.), marked, and released, at less than 7 days of age
were captured in ME-baited traps. In contrast, several other studies (Steiner 1952;
Steiner and Lee 1955; Wong et al. 1989; Shelly et al. 2008) reported young males
(5 days of age) showed relatively high response to ME. Wong et al. (1989), for
example, found that nearly 50 % of males responded to ME before their age of first
mating (13 days). Collectively, these studies involved different strains, different
procedures, and different test conditions, making it impossible to draw a robust
conclusion. Resolution is far from an arcane academic exercise, however, as
40 K.H. Tan et al.

knowledge of age-dependent response to ME is critical to predicting the success of
male annihilation efforts (see Barclay and Hendrichs, Chap. 11, this volume).
In contrast to intraspecific, age-dependent variation, little attention has been
given to interspecific differences in attraction to ME. In the most comprehensive
study to date, Wee et al. (2002) monitored the response of males of three closely
related Bactrocera species to serial dilutions of ME in small laboratory cages as
well as consumption of ME from microcapillary pipettes. The two assays yielded
the same trend, i.e., in decreasing order, the level of ME sensitivity was
B. dorsalis B. papayae B. carambolae. Most notably, males of B. carambolae
showed relatively weak attraction to the lure: the ME dose required to elicit
response of 50 % of B. carambolae males was 9 and 17 times higher than that
observed for B. papayae and B. dorsalis, respectively. Given the importance of ME
in trapping programs, additional studies of this type are clearly needed to better
characterize the detection sensitivity of area-wide trapping grids.
3.2.2 Fluorinated Analogs of Methyl Eugenol
One of the potential problems with the use of ME for fruit fly control is the reported
carcinogenicity of this compound in mice and rats (Miller et al. 1983) and microbes
(Schiestl et al. 1989; Sekizawa and Shibamoto 1982). ME has also been shown to
form DNA adducts in cultured human cells and thus may contribute to human
carcinogenesis (Zhou et al. 2007). However, several reviews (Smith et al. 2002;
Robinson and Barr 2006) conclude that ME does not pose a significant cancer risk
in humans, primarily because ME exposure in humans is as much as 1,000 times
below the level utilized to produce hepatic carcinoma in rats. Human subjects fed
approximately twice the daily average intake of safrole (a phenylpropene related to
methyl eugenol) over a 2 year period showed no carcinogenetic symptoms (Long
et al. 1963). In addition to the low exposure, ME in human blood serum is rapidly
eliminated and excreted (Schecter et al. 2004). ME may, in fact, have some benefits
to human health, e.g., reduction of cerebral ischemic injury (Choi et al. 2010) as
well as anti-anaphylactic properties (Kim et al. 1997). ME is a regular component
of the human diet (e.g., flavoring in baked goods and candy, Smith et al. 2002) and
is found in most spices and some plants, particularly in the family Lamiaceae, e.g.,
Ocimum basilicum (sweet basil) and O. sanctum (holy basil), which have high ME
contents and are regularly consumed as vegetables or used for culinary and medic-
inal purposes in Southeast Asian countries (Tan and Nishida 2012).
The fear of ME carcinogenicity and hepatoxicity to human health, whether
legitimate or a case of overreaction, has prompted some fruit fly scientists to search
for ‘safer’ alternative attractants for ME-responsive Bactrocera species and eval-
uate various phenylpropanoids with structural similarities to ME (Khrimian
et al. 1993, 1994, 2006, 2009; Liquido et al. 1998; Metcalf et al. 1975; Mitchell
et al. 1985). Two such analogs of ME are 4-[(2E)-3-fluoroprop-2-en-1-yl]-1,2-
dimethoxybenzene (FME), an analog fluorinated at the terminal carbon of the ME
side chain, and 1-fluoro-4,5-dimethoxy-2-(prop-2-en-1-yl)benzene (RFME), an

analog fluorinated at the 4 position of the ME aromatic ring. In field tests, FME was
as attractive to B. dorsalis males as ME (Khrimian et al. 1994), while RFME was
only about 50 % as attractive as ME (Khrimian et al. 2009). The good performance
of FME in field bioassays (Khrimian et al. 1994, 2006, 2009; Liquido et al. 1998)
showed that this compound is not only equally attractive to B. dorsalis but has an
added value as a more persistent lure. The carcinogencity of the terminal carbon
fluorinated compound has not yet been determined, but, if negative, FME could
serve as an excellent replacement for ME in trapping programs.
Jang et al. (2011) synthesized two additional fluorinated ME analogs,
1-(3,3-difluoroprop-2-en-1-yl)-2-fluoro-4,5-dimethoxybenzene, a ME analog
trifluorinated at the 4 position of the aromatic ring and at the terminal carbon of
the side chain, and 1-fluoro-2-(3-fluoroprop-2-en-1-yl)-4,5-dimethoxybenzene, a
ring and side-chain difluorinated analog. Although B. dorsalis males were attracted
strongly to and fed on the trifluoroanalog and difluoroanalog in a cage experiment,
field attractiveness of male oriental fruit fly to both was markedly lower than to
ME. In field bioassays, traps baited with difluoroanalog captured roughly 50 % as
many flies as traps baited with ME, while the trifluoroanalog captured only about
10 % as many males. Thus, di- or tri-fluorinated ME are likely not viable replace-
ments for ME as attractants for B. dorsalis and related species.
3.2.3 Plant Phenylpropanoids, Dimethoxycinnamyl Analogs
as Parapheromones of ME-Responsive Species
E-3,4-dimethoxycinnamyl alcohol and E-3,4-dimethoxycinnamyl acetate from
Spathiphyllum cannaefolium Schott (Araceae) (Chuah et al. 1996) and Hawaiian
lei flower, Fagraea berteriana A. Gray ex Benth. (Loganiaceae) were characterized
as attractants for B. dorsalis (Nishida et al. 1997). Although these compounds are
less volatile than ME, the feeding stimulant activity of the former was as high as
that of ME (Nishida et al. 1997). However, they will not replace ME in trapping of
B. dorsalis because of their low volatility and attractancy.
3.2.4 Raspberry Ketone, Raspberry Ketone Formate, and Cue Lure
As noted above, among lure-responsive Bactrocera species, the majority is
attracted to RK/CL. RK (Fig. 2.5) is found naturally as a fungal metabolite (Ayer
and Singer 1980) and in many plants besides raspberries (Rubus idaeus L.),
including other species in Rosaceae, Asteraceae, and Lamiaceae (formerly
Labiatae) (Hirvi et al. 1981; Hirvi and Honkanen 1984; Lin and Chow 1984;
Marco et al. 1988) as well as Orchidaceae (Nishida et al. 1993; Tan and Nishida
2005; Tan 2009). Drew (1974) reported that RK was developed as a male attractant
for B. tryoni in Australia in 1959 but provided no additional information regarding
the nature of this discovery. At approximately the same time, the United States
Department of Agriculture (USDA) was engaged in a large-scale screening of
42 K.H. Tan et al.

thousands of chemicals as potential fruit fly baits (Beroza and Green 1963). Based
on this screening process, Barthel et al. (1957) reported attraction of B. cucurbitae
males to anisylacetone (4-(4-methoxyphenyl)-2-butanone), a synthetic aromatic
ketone. In turn, Beroza et al. (1960), through continued testing of compounds
related to anisylacetone, synthesized CL (4-(4-acetoxyphenyl)-2-butanone),
which was a much more potent attractant for B. curcubitae males and is now
used worldwide in detection efforts for this species and other RK/CL-responsive
Bactrocera species (Jang et al. 2007). CL has not been isolated as a natural product
but is hydrolyzed to RK (also known as rheosmin; 4-(4-hydroxyphenyl)-2-
butanone), which as noted above is widespread in nature (Metcalf 1990; Metcalf
and Metcalf 1992b). In field tests, CL is a more potent attractant than RK (Alex-
ander et al. 1962; Keiser et al. 1973), likely owing to its high volatility relative to
that of RK (approximately 20 times greater, Metcalf 1990).
At this juncture, it is pertinent to point out that Nishida, Howcroft and Tan
(unpublished data) recently detected anisylacetone and CL (hitherto, not known as
natural products as mentioned above) in certain bactrocerophillous orchid flowers
(Bulbophyllum spp.) found in Papua New Guinea. They also showed that both the
compounds have differential attraction against RK-sensitive Bactrocera species in
field capture of wild males – with significantly more B. cucurbitae and
B. triangularis (Drew) captured in RK- than anisylacetone-baited traps and vice
versa for B. atramentata (Hering), B. bryoniae (Tryon) and B. frauenfeldi (Schiner)
(unpublished data). This shows that anisylacetone and CL, along with RK and
zingerone (see below), in nature may (a) play an important evolutionary role in the
Bactrocera fruit fly-orchid interactions, and (b) affect trapping of wild flies in
surveillance and quarantine detection for an areawide IPM/SIT program.
Although quantitative data are scant, it is generally accepted that CL is a weaker
attractant than ME (Cunningham 1989; Jang and Light 1996). Data from a mark-
release-recapture study (Shelly and Nishimoto 2011) conducted in Hawaii and
California confirmed this notion. For example, among flies released 100 m from
the lure source, 1–19 % of B. dorsalis males were captured in an ME-baited trap
compared to only 0.4–1.2 % of B. cucurbitae males captured in a CL-baited trap.
Correspondingly, with 5 ME- and 5 CL-baited traps per 2.59 km2
(operational
density in California, for example), there would be near certainty (99.9 %) of
detecting incipient B. dorsalis populations as small as 50–162 males, whereas the
same likelihood of detection for B. cucurbitae would require 310–350 males in the
population.
Although less potent, CL has been used in the same ways as ME, i.e.,
(i) detection and surveillance of invasive species (Gonzalez and Troncoso 2007;
Jessup et al. 2007), quarantine surveys and delimitation (Allwood 2000), suppres-
sion and eradication (Matsui et al. 1990; Vargas et al. 2000; Sookar et al. 2008), and
ecological studies, including faunal surveys (Osborne et al. 1997; Allwood 2000),
population dynamics and phenology (Itô et al. 1974; Harris et al. 1986; Vargas
et al. 1990), and dispersal (Fletcher 1989; Vargas et al. 1989; Kohama and Kuba
1996; Peck et al. 2005). As noted above, existing reviews address many of these
topics (see, in particular, Vargas et al. 2010a), and here we briefly address two

topics, namely (i) age-dependent variation in response to ME and (ii) comparative
performance of CL and raspberry ketone formate.
As with the B. dorsalis-ME association, attraction of B. cucurbitae males to CL
is related to sexual maturation, and findings have been inconsistent regarding the
level of response displayed by very young males. In particular, whereas Beroza
et al. (1960) observed attraction of newly emerged B. cucurbitae males to CL, other
studies (Monro and Richardson 1969; Fletcher 1974; Wong et al. 1991) report no
attraction until males are at least several days old. In the most comprehensive study,
Wong et al. (1991) found that wild B. cucurbitae males did not respond to CL until
10 days of age and that the timing of CL response and mating activity were highly
correlated. Based on this finding, these authors concluded that male annihilation
would be less effective against B. cucurbitae than B. dorsalis, because the closer
coincidence of lure response and sexual maturation in the former than the latter
means that fewer B. cucurbitae males would be killed in lure-baited traps prior to
mating than would be the case for B. dorsalis.
In attempting to identify a more potent lure than CL, several studies have
investigated the attractancy of the formate ester of RK, formic acid 4-(3-oxobutyl)
phenyl ester (RKF). In the early 1990s, Metcalf and Mitchell (1990) and Metcalf
and Metcalf (1992a, b) showed that RKF was more attractive to B. cucurbitae males
than either RK or CL. Despite this finding, no further research on RKF was
undertaken for about a decade, apparently because of concern regarding the rapid
hydrolytic conversion of RKF to RK (which, as noted above, is less volatile and less
attractive than CL, which hydrolyzes to RK at a slower rate, Beroza et al. 1960).
However, subsequent work (Casaña-Giner et al. 2003a, b) showed that rate of
hydrolysis of RKF to RK was likely overestimated. Furthermore, field testing
(Casaña-Giner et al. 2003a, b; Oliver et al. 2004; Jang et al. 2007) showed that
RKF-baited traps generally captured more B. cucurbitae males than CL-baited
traps. Additional field data, however, have not corroborated this result. Working
with B. cucurbitae, Vargas et al. (2010c) reported no difference in the catch of traps
baited with CL or RKF embedded in a biologically inert, waxy matrix (SPLAT),
and Shelly et al. (2012a) reported that traps baited with liquid CL had significantly
higher captures than traps baited with RKF presented as a liquid or in a polymeric
dispenser. Reasons for these inconsistent results are unknown, though it is possible
that variation in abiotic factors, which affected the conversion of CL and RKF to
RK, is responsible.
RKF has also been found to attract many other RK-responsive Bactrocera
species. Preliminary tests (Jang et al. unpublished) in Australia showed that RKF
plugs recaptured 1.5 times more sterile male Queensland fruit flies compared to a
CL plug. In an unpublished survey, Jang and colleagues found 19 Bactrocera
species in traps baited with CL and RKF in the northern territories of Australia.
Most of the species responded equally to either CL or RKF, but a few showed
higher trap captures to RKF than CL. The results from trap evaluations in the
Northern Cape York Peninsula and the Torres Strait showed that RKF had higher
trap captures of B. frauenfeldi, Bactrocera peninsularis (Drew and Hancock), and
Bactrocera neohumeralis (Hardy) compared to the CL plug.
44 K.H. Tan et al.

3.2.5 Presentation of ME and RK/CL
In the early 1980s, a proprietary product of International Pheromones Ltd was
marketed as ‘dorsalure’ (a mixture, in unknown proportions, of ME and CL) in
order to capture males of both ME- and RK/CL-sensitive species. In a species
survey conducted in five different ecosystems in Penang Island, Malaysia, it was
shown that CL and ME traps caught five RK-responsive and two ME-responsive
species, respectively, while ‘dorsalure’ traps caught only two RK- and one
ME-responsive species of the seven total species (Tan and Lee 1982). In addition,
Tan (1983) tested combinations of ME and CL (three liquid mixtures (v:v) of 2:1,
1:1 and 1:2) in the same trap, and all blends caught significantly fewer males of two
ME-responsive species – B. dorsalis and B. umbrosa – when compared with
ME-only baited traps. Thus, CL appeared to have caused a slight interference in
the male olfactory system of the ME-responsive species. More studies (Hooper
1978; Vargas et al. 2000; Shelly et al. 2004) have corroborated a reduction in
ME-responsive species with bait mixtures of ME and CL. Data regarding effects on
RK/CL responding species are inconsistent, however, as ME/CL blends have been
found to increase (Taiwanese data, cited by Hooper 1978), decrease (Hooper 1978),
or have no effect (Vargas et al. 2000) on catch numbers of RK/CL sensitive species.
In several large-scale detection programs (e.g., California, USA), Bactrocera
lures are applied as liquids to cotton wicks, which are then placed in Jackson traps.
To minimize worker risk owing to inadvertent spillage and exposure, field tests,
conducted primarily in Hawaii, have compared the efficacy of the standard liquid
formulation with different solid dispensers containing ME and CL separately or in
combination in the same device. In general, studies (Hiramoto et al. 2006; Suckling
et al. 2008; Jang 2011; Jang et al. 2013; Vargas et al. 2009, 2010b; Shelly 2010b;
Shelly et al. 2011a, b; Leblanc et al. 2011) have shown that the solid dispensers
perform as well as or even better than the liquid application (but see Wee and Shelly
2013 for an exception). Interestingly, two studies (Vargas et al. 2012; Shelly
et al. 2012b) conducted in Hawaii further reported that traps baited with solid
wafers containing ME, RK, and TML captured as many males of B. dorsalis,
B. cucurbitae, and C. capitata as traps baited with a single lure in liquid form.
The use of such triple-lure dispensers holds promise, not only in reducing worker
safety, but also in reducing costs of trapping supplies and trap monitoring and
servicing.
3.2.6 Zingerone
Zingerone (4-(4-hydroxy-3-methoxy-phenyl)-2-butanone, 4-hydroxy-3-
methoxybenzyl- acetone, vanillylacetone) (Fig. 2.5) is a phenylbutanoid responsi-
ble for the pungency of ginger, Zingiber officinale (L.) H. Karst. Field studies
showed that zingerone present in flowers of Bulbophyllum patens King and
B. baileyi F. Muell. attracted males of both ME- and RK-responsive Bactrocera

species, particularly, B. dorsalis and B. cucurbitae/B. albistrigata (Tan and Nishida
2000, 2007). Because of the presence of a hydroxyl group and a butanone side chain
(both are also found in RK) as well as a methoxy (found in ME) moiety attached to
the benzene ring (Fig. 2.5), zingerone attracts males of both ME- and
RK-responsive Bactrocera species, albeit relatively very weak attraction in com-
parison to ME and RK (Tan and Nishida 2007). Zingerone, when consumed by
B. dorsalis males, is converted to zingerol, attractive to conspecific females, as a
component of male sex pheromone (Tan and Nishida 2007). However, in
B. cucurbitae males, zingerone is sequestered largely unchanged into the rectal
gland (Nishida et al. 1993). Khoo and Tan (2000) and Kumaran et al. (2013) have
further examined the effects of male feeding on zingerone on their success in
attracting mates and obtaining matings in B. cucurbitae and B. tryoni, respectively.
After the discovery of floral zingerone attracting both ME and RK-responsive
species (Tan and Nishida 2000, 2007), Fay (2010) explored the structure-activity
relationships of 50 different phenylpropanoids and phenylbutanoids that might
attract the non-responsive Bactrocera and Dacus to the two potent attractants. It
was shown that certain non-responsive Bactrocera species, namely B. aglaiae
(Hardy), B. aurea (May), and B. speewahensis Fay and Hancock (a new species),
as well as a rarely trapped Dacus secamoneae Drew, were captured only in traps
baited with zingerone and not in ME and RK/CL traps (Fay 2010). Further, a
qualitative field evaluation using traps baited with zingerone, RK/CL, or ME
conducted in north-eastern Australia showed that Bactrocera jarvisi (Tryon), pre-
viously known to be attracted to RK/CL, was strongly attracted to zingerone, with
more than 97 % of flies of this species captured in traps baited with the attractant. In
contrast, B. neohumeralis and B. tryoni males were caught more frequently in RK
traps (Fay 2012). In north Queensland, B. jarvisi invariably constituted 97–99 % of
the total catch, and zingerone is “now starting to be used in various places around
the country (Australia) for both detection and male annihilation purposes” (Harry
Fay 2012 – personal communication). These very interesting results certainly
suggest that zingerone should be tested more widely throughout the Asia-Pacific
region for possible attraction of other non-responsive Bactrocera species (which
constitute approximately 50 % of the total Bactrocera species) to the commonly
used ME and RK/CL attractants.
3.2.7 α-Ionone Analogs for Bactrocera latifrons
α-Ionol (latilure) and its analogs (Fig. 2.5) were found as attractants for trapping
males of the solanaceous fruit fly, B. latifrons, which shows no affinity to either ME
or CL (Flath et al. 1994a). Although the attractiveness of α-ionol is much lower than
that of ME and CL for B. dorsalis and B. cucurbitae, respectively, cade oil and its
ingredients (e.g., eugenol) synergistically enhanced the attraction (McQuate and
Peck 2001; McQuate et al. 2004, 2008a, b). On the contrary, isophorone (3,5,5-
trimethyl-2-cyclohexene-1-one) and isophorol mixed with α-ionol attracted more
males than the respective individual compounds (Ishida et al. 2008). Furthermore, a
46 K.H. Tan et al.

series of 3-oxygenated α-ionone analogs have been found as more potent attrac-
tants/phagostimulants than α-ionone/ionol (Ishida et al. 2008; Nishida et al. 2009;
Enomoto et al. 2010). These C13-norterpenoid analogs, resembling raspberry
ketone-type phenylbutanoid structure, are present in various fruit tissues (mostly
in glycosidic-forms). Ingested 3-oxgenated α-ionones by B. latifrons males were
selectively biotransformed to a variety of derivatives, which were eventually
sequestered into the rectal gland – suggesting a possible role as sex pheromone,
although the actual biological function is still unknown (Nishida et al. 2009;
Enomoto et al. 2010).
3.3 Ceratitis
The history surrounding the development of male lures for C. capitata has been
recounted numerous times (Chambers 1977; Cunningham 1989; Millar 1995; Jang
and Light 1996), and no purpose is served in repeating it here. Instead, we focus on
a few selected topics, namely (i) α-copaene and natural oils as male attractants and
(ii) the chemical characterization and modification of trimedlure (TML).
3.3.1 α-Copaene and Natural Oils as Male Attractants
Ripley and Hepburn (1935) first described the attraction of male Ceratitis, in
particular males of C. rosa, to angelica seed oil (Angelica archangelica (Linn.)).
Steiner et al. (1957) later reported the attraction of C. capitata males to the seed oil,
which was used intensively in the 1956 Florida campaign against C. capitata to the
point of exhausting the world supply. Over a decade later, two researchers
(Fornasiero et al. 1969; Guiotto et al. 1972) identified α-copaene as the main
attractant in angelica seed oil and α-ylangene as a secondary attractant. In a series
of field trials in Hawaii, α-copaene was found to be more attractive to medfly males
than TML (Flath et al. 1994b, c). In addition, the stereochemistry of this compound
was critical in determining its potency as even slight deviations from the
dextrorotary form ((+)-α-copaene) led to decreased attractiveness. Although data
are not provided and the enantiomer is not identified, α-copaene was reported to be
2–5 times more attractive to male medflies than TML in field tests (Cunningham
1989).
While highly attractive, (+)-α-copaene has limited practical use, because its
synthesis is extremely difficult and expensive and its concentration in most natural
(plant) sources is low. Methods for synthesizing (+)-α-copaene have been devel-
oped (Heathcock 1966; Heathcock et al. 1967; Corey and Watt 1973), but these are
laborious and yield only small amounts. Millar (1995) noted that new synthetic
pathways have been developed for copaene isomers (Kulkarni et al. 1987; Wenkert
et al. 1992), thus opening the possibility that simpler, and more easily synthesized,
analogs might be identified as practical alternatives. Regarding plant sources, where

α-copaene occurs, the levorotatory isomer usually predominates, and in those
instances where (+)-α-copaene dominates, the overall content of copaene is typi-
cally low (1 % of the essential oil prepared from the plant – Takeoka et al. 1990).
Angelica seed oil differs from most plant species sampled (Buttery et al. 1985;
Elzen et al. 1985) in that (+)-α-copaene appears to be the more common stereoiso-
mer and to be relatively abundant (0.16–0.34 % of commercial angelica seed oil)
(Jacobson et al. 1987).
Another commercially available, natural product, ginger root oil, also contains
relatively high amounts of (+)-α-copaene and has been investigated as a medfly
attractant. A distillation procedure has been developed that increases the concen-
tration of (+)-α-copaene from 0.4 % in commercially available oil to 8 % in the
enriched oil (Shelly and Pahio 2002). However, when applied as a paste at varying
doses to cotton wicks, traps baited with the enriched ginger root oil captured in
significantly fewer C. capitata males than traps baited with liquid TML (Shelly and
Pahio 2002). Moreover, the enriched oil appeared to lose its potency rather quickly:
paste aged 5 days resulted in 10–20 % fewer captures than fresh paste. A second
study (Shelly 2013) also conducted in Hawaii confirmed the greater attractancy of
TML plugs to enriched ginger root oil applied in liquid form. In contrast,
Mwatawala et al. (2013), working in Tanzania, found that trap captures with
enriched ginger root oil were equal to or greater than those with TML for four
Ceratitis species (including the medfly) and that the oil captured males of one
species (C. cosyra not typically found in TML-baited traps). Based on these results,
the authors suggest that enriched ginger root oil is a viable alternative to TML in
Ceratitis detection programs in Africa.
The discrepancy in the results for the medfly between Hawaii and Africa could
reflect differences in the composition of the oils used in the two regions. In a study
of avocado varieties, Niogret et al. (2011) found that the behavioral and EAG
responses of medflies were not directly related to the amount of α-copaene in the
volatiles of the different varieties. For example, α-copaene comprised 31 % of the
sesquiterpenes for one of the least attractive varieties but only 12 % for the most
attractive variety. Thus, the presence and concentration of sesquiterpenes other than
α-copaene may affect medfly response to natural oils, and variation in the chemical
composition of ginger oils from different suppliers could generate different results
in trapping studies.
3.3.2 Trimedlure
Since its discovery approximately 50 years ago (Beroza et al. 1961), TML (tert-
butyl 4(and 5)-chloro-trans 2-methylcyclohexane-1-carboxylate) has become
widely, but not universally (as noted below), adopted as the chief male attractant
used in detection and surveillance programs for C. capitata (Jang et al. 2001). These
authors recognized TML to be a mixture of isomers, with four isomers
predominating (Beroza and Sarmiento 1964), but complete resolution of the iso-
meric constitution of trimedlure was not achieved until Leonhardt et al. (1982)
48 K.H. Tan et al.

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